NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways
Abstract
:1. Introduction
2. Materials and Methods
2.1. Animals
2.2. Mammalian Cell Culture—Cell Lines
2.3. Generation of NanoFIRE and Other Expression Constructs
2.4. Lentivirus Production and Generation of Stable Cell Lines
2.5. NanoLuciferase Reporter Assays—Stable Cell Lines
2.6. Primary Granulosa Cell Culture and NanoLuciferase Reporter Assays
2.7. High Content Imaging of Fluorescent Reporter Cells
2.8. Statistical Analysis and Figures
3. Results
3.1. Design and Characterisation of HRE-NanoFIRE to Investigate HIF Signalling
3.2. HRE-NanoFIRE Displays Robust Reporter Response to Hypoxia and Hypoxia Mimetics
3.3. HRE-NanoFIRE Is an Effective HIF Reporter System in Diverse Cell Lines and Primary Cells
3.4. HRE-NanoFIRE Is More Sensitive Than Equivalent Fluorescent Reporter Systems
3.5. NanoFIRE Can Be Adapted to Investigate Transcriptional Regulators and Synthetic Transcription Factors
3.6. NanoFIRE Can Be Adapted to Investigate Other Transcription Factors
4. Discussion
Author Contributions
Funding
Institutional Review Board Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Roennfeldt, A.E.; Allen, T.P.; Trowbridge, B.N.; Beard, M.R.; Whitelaw, M.L.; Russell, D.L.; Bersten, D.C.; Peet, D.J. NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways. Biomolecules 2023, 13, 1545. https://doi.org/10.3390/biom13101545
Roennfeldt AE, Allen TP, Trowbridge BN, Beard MR, Whitelaw ML, Russell DL, Bersten DC, Peet DJ. NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways. Biomolecules. 2023; 13(10):1545. https://doi.org/10.3390/biom13101545
Chicago/Turabian StyleRoennfeldt, Alison E., Timothy P. Allen, Brooke N. Trowbridge, Michael R. Beard, Murray L. Whitelaw, Darryl L. Russell, David C. Bersten, and Daniel J. Peet. 2023. "NanoFIRE: A NanoLuciferase and Fluorescent Integrated Reporter Element for Robust and Sensitive Investigation of HIF and Other Signalling Pathways" Biomolecules 13, no. 10: 1545. https://doi.org/10.3390/biom13101545